Aminoacyl-tRNA synthetases are required for accurate charging of tRNA. The tyrosyl-tRNA synthetase (tyrS) gene in Bacillus subtilis is a member of the T box family of tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria. Genes in this group (27 genes to date) contain transcriptional termination signals in the mRNA leader region, upstream of the start of the coding sequence. Readthrough of the terminator is induced by interaction of the cognate uncharged tRNA with the leader, which promotes formation of an antiterminator structure, preventing terminator formation. The T box mechanism appears to be specific for Gram-positive transcription regulation and cannot be deciphered from studies in E. coli. The goal of this proposal is to characterize the biochemistry of transcription antitermination of the tyrS gene in Bacillus subtilis. tRNA directed readthrough of the B. subtilis tyrS leader region terminator does not occur in E. coli or in vitro under standard conditions. Observations from this lab and others suggests the presence of an unidentified factor(s) in B. subtilis that is necessary for appropriate terminator/antiterminator formation or stability. Working with an in vitro transcription system, we hope to identify additional factors or conditions which promote readthrough. In vivo, varied growth conditions, leader region mutants and trans-acting mutants will be examined. The effects of NusA, a potential regulatory factor, will be evaluated. The kinetics of transcription of the tyrS leader region will be investigated, enabling the examination of the dynamics of the T box transcription mechanism.